Porcine circovirus (PCV) was originally identified as a contaminant of porcine kidney cell cultures (PK15 ATCC CCL-33). The PCV virion has been characterized as being an icosahedral, non-enveloped virus with a single-stranded circular DNA of about 1.76 kb. PCV was classified in the genus Circovirus of the Circoviridae family, which consists of other animal circoviruses such as psittacine beak-feather disease virus, goose circovirus, canary circovirus, and pigeon circovirus. Two genotypes of PCV have been recognized. The PK15 cell-derived PCV has been considered to be nonpathogenic to pigs, and is designated PCV type 1 (PCV1). On the other hand, PCV type 2 (PCV2) has been accepted as the major infectious agent involved in several pig diseases. PCV2 associated diseases cause significant economic losses to swine producers worldwide. PCV2 associated diseases are described in WO2007/076520 and include, for example, Postweaning Multisystemic Wasting Syndrome (PMWS), Porcine Dermatitis and Nephropathy Syndrome (PDNS), Porcine Respiratory Disease Complex (PRDC), reproductive disorders, granulomatous enteritis, exudative epidermitis, necrotizing lymphadenitis, and congenital tremors. Occurrences of PCV2 subtype A (PCV2A) and PCV2 subtype B (PCV2B) have been reported particularly in 2000 in West Europe and in Central Europe in 2003. More recently similar changes have been reported in 2008 in wild boars.
Currently developed PCV2 vaccines, such as Circovac® (Merial), Ingelvac®, CircoFLEX (Boehringer lngelheim Vetmedica), or Suvaxyn®, are either inactivated PCV2 vaccines or Sub-Unit vaccines. Regarding inactivated PCV2 vaccines, current PCV2 strains subtype A or B present several weaknesses. Particularly, PCV2 viruses can only be produced at low titers, generally less than 105 TCID50 viral particles per ml. Also, these viruses cannot be maintained in tissue cultures and permanently infected cell lines. Regarding PCV2 Sub-Unit vaccines, they typically use a purified, recombinant PCV2 capsid protein produced by expression of the ORF2 gene of PCV2 in a baculovirus system. In this regard, the protein encoded by ORF2 of PCV2 isolates Imp1011 has been reported in EP1741785. A protein encoded by ORF2 of PCV2 isolate PCV2Rm has been reported in WO2010/061000. The protein encoded by ORF2 of PCV2 isolate 412 has been reported in EP1816200. Another protein encoded by an ORF2 of a further PCV2 isolate has been reported in EP1036180 or EP2225367.
Expression efficiency and immunogenicity of these natural capsid proteins are, however, not optimal and do not always provide the required level of immune protection in vaccinated animals. In particular, the ORF2 protein comprises a nuclear localization sequence which leads to expression of the protein in the nucleus of the cells. Such intracellular localization does not facilitate extraction or purification of the protein and could also prevent or reduce the effectiveness of DNA or vector vaccines which express the ORF2 protein in vivo in the animals.
WO2010/068969 proposes to modify the expression profile of ORF2 to express an ORF2 antigen in soluble form by using foreign secretion signal peptide sequences. In this application, it is proposed to fuse ORF2 to a secretion signal or to a cell membrane signal, and to include in the construct a cleavage site so that the soluble ORF2 can be released in soluble form. This application proposes a long prophetic list of potential candidate secretory peptides. However, the application does not contain any experimental data showing that effective or improved expression/immunogenicity may be obtained by modifying the expression profile of an ORF2. No construct is disclosed allowing effective immunization.
The present invention proposes novel improved constructs for expressing antigenic polypeptides. The present invention discloses fusion products that are specifically adapted for the improved expression of antigenic polypeptides at the cell surface, especially using a viral expression vector such as a swinepox virus.
The invention shows that by expressing an antigenic polypeptide at the surface of infected/transduced cells using a B5R-derived addressing signal, an improved immune response is obtained, causing effective protection. By presenting the antigen at the cell surface in vivo in the animal, the vaccines of the invention most effectively deliver and expose the antigen to the immune system, particularly to immune cells such as lymphocytes, dendritic cells and macrophages. By presenting the antigen at the cell surface, the invention provides the immunogen in an active conformation to elicit a potent protective immune response. The invention may be applied to any antigenic polypeptide, particularly viral antigens.